Comparative analysis of expressed sequence tags (ESTs) between normal group and softness syndrome group in Halocynthia roretzi

To identify the cause of mass mortality in ascidians, we constructed cDNA libraries of both the normal and softness syndrome group in Halocynthia roretzi. To perform comparative analysis of transcripts between the two groups, we sequenced about 1,000 random clones. All the sequences obtained from the clones were processed to remove the vector region and low quality sequences through base calling and vector trimming. We collected 906 sequences with average length of 463 bp in the normal group and 1014 sequences in the softness syndrome group with an average length of 696 bp. Clustering and assembling of EST sequences using TGICL package resulted in 906 distinct sequences composed of 517 singletons and 77 contigs in 75 clusters in normal group and 1014 distinct sequences composed of 707 singletons and 120 contigs in 120 clusters in the softness syndrome group. All sequences derived from the two groups were compared against the NCBI Non-redundant database using BLASTX algorithms. As a result, 493 sequences in the normal group and 861 sequences in the softness syndrome group had significant hits within the database. In addition, we listed genes that showed differential expression in the softness syndrome group. Transcript levels of calponin increased by 11-fold and both E3 ubiquitin-protein ligase MARCH3 and selenium dependent salivary glutathione peroxidase by 5-fold in the softness syndrome group. Also, the expression of four genes including muscle actin increased by 4-fold. In contrast, we observed down-regulation of genes encoding trypsinogen 1, cathepsin D protein, serine protease, and halocidin precursor, decreasing by more than 6-fold. Herein, we identified differential expression of genes involved in the contraction and regulation of muscle cells and immune reaction in H. roretzi with softness syndrome. This study is the first report on gene expression changes occurring in H. roretzi with softness syndrome and would be useful in further studies.